Glyceride cleavage is typically performed in industry using high-temperature steam treatment which does not discriminate between saturated and unsaturated fatty acid or fatty acid chain length. The use of enzymes, particularly lipases, as catalysts for efficient glyceride splitting at mild, low energy-demanding conditions would offer many economic advantages over these prior methods. The use of a selective lipase for the preparation of high purity oleic acid from fats or oils containing low levels of oleic acid would offer additional economic advantages over these prior methods.
A. Lipases, Generally
Lipases are a group of enzymes belonging to the esterases, and are also called glyceroester hydrolases or acylglycerol-acylhydrolases. These enzymes hydrolyze fat and oils (present in ester form, such as glycerides), yielding the base components of glycerol and fatty acids. These enzymes also catalyze digestion. Lipases are widely found throughout the plant and animal worlds, including molds, bacteria, milk and milk products, and animal tissues such as the pancreas. Lipases have been widely used to split fats without damaging sensitive constituents, such as vitamins or unsaturated fatty acids. (The Merck Index, p. 868).
Lipases generally have two types of specificity, positional specificity and fatty acid specificity. Positional specificity or regiospecificity permits some lipases to split the ester bonds at particular positions on the triglyceride backbone. Aspergillus niger lipases display this type of specificity. Hata et al., J. Biochem. 86:6, 1821-1827 (1986). Pancreatic lipase and Pseudomonas fragi lipase reportedly attack triglycerides at the 1-, 3-positions. Alford et al., JAOCS 42, 1038-40 (1961). An ideal lipase for total fat splitting would have the following properties:
1) broad substrate specificities, PA1 2) no significant interferences, PA1 3) high specific activity, PA1 4) a degree of hydrolysis of 100%. PA1 a.) culturing Geotrichum candidum NRRL Y552 in a nutrient medium, e.g. nutrient agar; PA1 b.) exposing the cultured strain to ultraviolet light for a sufficient period of time to induce mutation; PA1 c.) incubating the resulting mutants with agitation in a nutrient broth; PA1 d.) freezing the suspension; PA1 e.) thawing the suspension, and PA1 f.) repeating steps a.) through e.), yielding the desired organism. PA1 A. Biocryl BPA-1000 cation exchange chromatography to clarify the cell-free broth of nonproteinaceous material; PA1 B. Concentration of the UNLipase by ethanol precipitation followed by centrifugation; PA1 C. Resuspension of the ethanol precipitation, followed by hydrophobic interaction chromatography on octyl-sepharose; PA1 D. Chromatography On Q-Sepharose with final concentration of the lipase by ultrafiltration using centricon 30 ultrafiltration units.
Fatty acid specificity is displayed by lipases which indiscriminately split all of the ester bonds. Certain amounts of fatty acid specificity have been found in some Geotrichum candidum lipases. Hata et al., J. Biochem. 86:6 1821-1827 (1986). These lipases are generally specific for certain chain length fatty acids or double bond positions. Osterberg et al., JAOCS, 66:9 (1989).
B. Prior Research on Geotrichum candidum Lipases
G. candidum is a mold with septate mycelia, found growing on sour cream and cheese in a firm white mass. Jensen, Lipids, 9, 149-157 (1974). It has long been known for its relatively selective lipases. While extensive research has been performed on G. candidum lipases, little success has been achieved with regard to obtaining a truly selective lipase.
A close examination of the prior art indicates that, while it has been known that G. candidum lipase has selectivity for oleic acid containing triglycerides over short chain fatty acids and saturated fatty acids, no lipase has attained the level of oleic acid selectivity shown by the UNLipase, which means Unsaturation-specific Lipase, enzyme of the present invention. Please see the following discussion of the prior art.